Optical imaging approach to study protein-DNA interaction and nuclear organization in cultured living cells

Subcellular trafficking and localization of the glucocorticoid receptor (GR) has been followed with the green fluorescent protein (GFP)-GR chimeric protein. When expressed in mammalian tissue culture cells, GFP is present throughout the cellular Volume but is excluded from some vesicles in the cytoplasm and nucleoli in the nucleus. When fused to GR, green fluorescence becomes restricted to the cytoplasm and concentrates along fibrillar structures with no green fluorescence detected in the nucleus. Treatment with ligand leads to translocation to the nucleus with agonist ligand, such as dexamethasone, inducing accumulation of GFP-GR chimeric protein into nuclear foci. Accumulation into foci, in part, reflects targeting of GFP-GR to target genes, since the introduction of exogenous GR-binding sites leads to new patterns of accumulation. In the case of a cell line containing a large tandem array of the mouse mammary tumor virus long terminal repeat (MMTV LTR), the foci are not reserved leading to the appearance of a ribbon-like structure upon accumulation of GFP-GR on the GR-binding sites in the MMTV LTR tandem array. Mutational analysis of the GR DNA-binding domain reveals the importance of this domain for both the accumulation into nuclear foci as well as targeting to the GR-binding sites in the large tandem array. The use of GFP chimeric proteins and large tandem array of a define DNA sequence provides a unique opportunity to study DNA-protein interaction and the organization of the interphase nucleus in live cells.